Small-Molecule Induction Promotes Corneal Endothelial Cell Differentiation From Human iPS Cells

Purpose: Corneal endothelial cells (CECs) serve as a barrier and foothold for the corneal stroma to maintain the function and transparency of the cornea. Loss of CECs during aging or disease states leads to blindness, and cell replacement therapy using either donated or artificially differentiated CECs remains the only curative approach.Methods: Human induced pluripotent stem cells (hiPSCs) that were cultured in chemically defined medium were induced with dual-SMAD inhibition to differentiate into neural crest cells (NCCs). A small-molecule library was screened to differentiate the NCCs into corneal endothelial-like cells. The characteristics of these cells were identified with real-time PCR and immunofluorescence. Western blotting was applied to detect the signaling pathways and key factors regulated by the small molecules.Results: We developed an effective protocol to differentiate hiPSCs into CECs with defined small molecules. The hiPSC-CECs were characterized by ZO-1, AQP1, Vimentin and Na+/K+-ATPase. Based on our small-molecule screen, we identified a small-molecule combination, A769662 and AT13148, that enabled the most efficient production of CECs. The combination of A769662 and AT13148 upregulated the PKA/AKT signaling pathway, FOXO1 and PITX2 to promote the conversion of NCCs to CECs.Conclusion: We established an efficient small molecule-based method to differentiate hiPSCs into corneal endothelial-like cells, which might facilitate drug discovery and the development of cell-based therapies for corneal diseases.

A small molecule reveals role of insulin receptor-insulin like growth factor-1 receptor heterodimers

The insulin receptor (IR) and insulin like growth factor-1 receptor (IGF-1R) are heterodimers consisting of 2 extracellular α-subunits and 2 transmembrane β-subunits. IR α/β and IGF-1R α/β hemi-receptors can heterodimerize to form hybrids composed of one IR α/β and one IGF-1R α/β. Widely distributed in mammalian tissues, in contrast to IR and IGF-1R the physiological function of hybrids is unclear. To identify tool compounds that inhibit hybrid formation we performed a high-throughput small molecule screen based on a homology model of hybrid structure. Our studies unveil a first in class quinoline-containing heterocyclic small molecule that reduces hybrids by >50% in human umbilical vein endothelial cells (HUVECs) with no effect on IR or IGF-1R. Downstream of IR and IGF-1R our small molecule led to reduced expression of the negative regulatory p85α subunit of phosphatidylinositol 3-kinase, an increase in phosphorylation of its downstream target Akt and enhanced insulin and shear-induced phosphorylation of Akt. We show that hybrids have a role in human EC physiology distinct from IR and IGF1R.

Pharmacologic rescue of circadian β-cell failure through P2Y1 purinergic receptor identified by small-molecule screen

The mammalian circadian clock drives daily oscillations in physiology and behavior through an autoregulatory transcription feedback loop present in central and peripheral cells. Ablation of the core clock within the endocrine pancreas of adult animals impairs the transcription and splicing of genes involved in hormone exocytosis and causes hypoinsulinemic diabetes. However, identification of druggable proteins and pathways to ameliorate the burden of circadian metabolic disease remains a challenge. Here, we generated beta cells expressing a nano-luciferase reporter within the proinsulin polypeptide to screen 2,640 pharmacologically-active compounds and identify insulinotropic molecules that bypass the secretory defect in clock mutant beta cells. We validated lead compounds in primary mouse islets and identified known modulators of ligand-gated ion channels and G-protein coupled receptors, including the antihelmintic ivermectin. Single-cell electrophysiology in circadian mutant mouse and human cadaveric islets validated ivermectin as a glucose-dependent secretagogue. Genetic, genomic, and pharmacologic analyses established that the molecular clock controls the expression of the purinergic P2Y1 receptor to mediate the insulinotropic activity of ivermectin. These findings identify the P2Y1 purinergic receptor as a target to rescue circadian beta-cell failure and establish a chemical genetic screen for endocrine therapeutics.

A small molecule screen identifies novel inhibitors of mechanosensory nematocyst discharge in Hydra

Cnidarians are characterized by the possession of stinging organelles, called nematocysts, which they use for prey capture and defense. Nematocyst discharge is controlled by a mechanosensory apparatus with analogies to vertebrate hair cells. Members of the transient receptor potential (TRPN) ion channel family are supposed to be involved in the transduction of the mechanical stimulus. A small molecule screen was performed to identify compounds that affect nematocyst discharge in Hydra. We identified several [2.2]paracyclophanes that cause inhibition of nematocyst discharge in the low micro-molar range. Further structure–activity analyses within the compound class of [2.2]paracyclophanes showed common features that are required for the inhibitory activity of the [2.2]paracyclophane core motif. This study demonstrates that Hydra can serve as a model for small molecule screens targeting the mechanosensory apparatus in native tissues.

High-throughput small molecule screen identifies inhibitors of microsporidia invasion and proliferation in C. elegans.

Microsporidia are a diverse group of fungal-related obligate intracellular parasites that infect most animal phyla. Despite the emerging threat that microsporidia have become to humans and agricultural animals, few reliable treatment options exist. To identify novel chemical inhibitors of microsporidia infection, we developed a high-throughput screening method using Caenorhabditis elegans and the microsporidia species Nematocida parisii. We screened the Spectrum Collection of 2,560 FDA-approved compounds and natural products to identify compounds that prevent C. elegans progeny inhibition caused by N. parisii infection. We developed a semi-automated method for quantifying C. elegans progeny number in liquid culture, confirming 11 candidate microsporidia inhibitors. We show that five compounds prevent microsporidia infection by inhibiting spore firing, and demonstrate that one compound, dexrazoxane, slows infection progression. Together, our results demonstrate the effectiveness of C. elegans as a model host for drug discovery against intracellular pathogens and provide a scalable high-throughput system for the identification and characterization of additional microsporidia inhibitors.

TLR7 ligation augments hematopoiesis in Rps14 (uS11) deficiency via paradoxical suppression of inflammatory signalling.

Myelodysplastic syndrome (MDS) is a haematological malignancy characterised by blood cytopenias and predisposition to acute myeloid leukaemia (AML). Therapies for MDS are lacking, particularly those that impact the early stages of disease. We developed a model of MDS using zebrafish using knockout of Rps14, the primary mediator of the anaemia associated with del (5q) MDS. These mutant animals display dose- and age-dependent abnormalities in haematopoiesis, culminating in bone marrow failure with dysplastic features. We utilized rps14 knockdown to undertake an in vivo small molecule screen to identify compounds that ameliorate the MDS phenotype, identifying imiquimod, an agonist of TLR7 and TLR8. Imiquimod alleviates anaemia by promoting haematopoietic stem and progenitor cell expansion and erythroid differentiation, the mechanism of which is dependent on TLR7 ligation and Myd88. TLR7 activation in this setting paradoxically promoted an anti-inflammatory gene signature indicating crosstalk between pro-inflammatory pathways endogenous to Rps14 loss and NFkappaB pathway via TLR7. Finally, we show that in highly purified human bone marrow samples from anaemic patients, imiquimod leads to an increase in erythroid output from myelo-erythroid progenitors and common myeloid progenitors. Our findings have both specific implications for the development of targeted therapeutics for del (5q) MDS and wider significance identifying a potential role for TLR7 ligation in modifying anaemia.

Small molecule inhibition of ATM kinase increases CRISPR-Cas9 1-bp insertion frequency

Mutational outcomes following CRISPR-Cas9-nuclease cutting in mammalian cells have recently been shown to be predictable and, in certain cases, skewed toward single genotypes. However, the ability to control these outcomes remains limited, especially for 1-bp insertions, a common and therapeutically relevant class of repair outcomes. Here, through a small molecule screen, we identify the ATM kinase inhibitor KU-60019 as a compound capable of reproducibly increasing the fraction of 1-bp insertions relative to other Cas9 repair outcomes. Small molecule or genetic ATM inhibition increases 1-bp insertion outcome fraction across three human and mouse cell lines, two Cas9 species, and dozens of target sites, although concomitantly reducing the fraction of edited alleles. Notably, KU-60019 increases the relative frequency of 1-bp insertions to over 80% of edited alleles at several native human genomic loci and improves the efficiency of correction for pathogenic 1-bp deletion variants. The ability to increase 1-bp insertion frequency adds another dimension to precise template-free Cas9-nuclease genome editing.

Small Molecule Screen Reveals Joint Regulation of Stress Granule Formation and Lipid Droplet Biogenesis

Metabolic regulation is a necessary component of all stress response pathways, because all different mechanisms of stress-adaptation place high-energy demands on the cell. Mechanisms that integrate diverse stress response pathways with their metabolic components are therefore of great interest, but few are known. We show that stress granule (SG) formation, a common adaptive response to a variety of stresses, is reciprocally regulated by the pathways inducing lipid droplet accumulation. Inability to upregulate lipid droplets reduces stress granule formation. Stress granule formation in turn drives lipid droplet clustering and fatty acid accumulation. Our findings reveal a novel connection between stress response pathways and new modifiers of stress granule formation.

Small-molecule screen reveals synergy of cell cycle checkpoint kinase inhibitors with DNA-damaging chemotherapies in medulloblastoma

Medulloblastoma (MB) consists of four core molecular subgroups with distinct clinical features and prognoses. Treatment consists of surgery, followed by radiotherapy and cytotoxic chemotherapy. Despite this intensive approach, outcome remains dismal for patients with certain subtypes of MB, namely, MYC-amplified Group 3 and TP53-mutated SHH. Using high-throughput assays, six human MB cell lines were screened against a library of 3208 unique compounds. We identified 45 effective compounds from the screen and found that cell cycle checkpoint kinase (CHK1/2) inhibition synergistically enhanced the cytotoxic activity of clinically used chemotherapeutics cyclophosphamide, cisplatin, and gemcitabine. To identify the best-in-class inhibitor, multiple CHK1/2 inhibitors were assessed in mice bearing intracranial MB. When combined with DNA-damaging chemotherapeutics, CHK1/2 inhibition reduced tumor burden and increased survival of animals with high-risk MB, across multiple different models. In total, we tested 14 different models, representing distinct MB subgroups, and data were validated in three independent laboratories. Pharmacodynamics studies confirmed central nervous system penetration. In mice, combination treatment significantly increased DNA damage and apoptosis compared to chemotherapy alone, and studies with cultured cells showed that CHK inhibition disrupted chemotherapy-induced cell cycle arrest. Our findings indicated CHK1/2 inhibition, specifically with LY2606368 (prexasertib), has strong chemosensitizing activity in MB that warrants further clinical investigation. Moreover, these data demonstrated that we developed a robust and collaborative preclinical assessment platform that can be used to identify potentially effective new therapies for clinical evaluation for pediatric MB.